RP-HPLC同时测定独活中蛇床子素和二氢欧山芹醇当归酸酯的含量

 目的 建立同时测定独活中蛇床子素和二氢欧山芹醇当归酸酯含量的反相高效液相色谱法。方法 Shiseido C₁₈ MG（4.6 mm×250 mm，5 μm）色谱柱，流动相乙腈-水(49∶51)，检测波长330 nm，流速1.0 mL·min^-1，柱温30 ℃。结果 蛇床子素在0.008 2~0.656 mg·mL^-1内呈良好线性，r=1.000 0，平均回收率99.6%（RSD=2%）；二氢欧山芹醇当归酸酯在0.003 6~1.536 mg·mL^-1内呈良好线性，r=0.999 9，平均回收率99.4%（RSD=2.2%）。结论 通过对22批不同来源独活药材与饮片的含量测定，证明该方法简单，准确可靠，重复性好。在重庆巫山地区以海拔1 600~1 700 m、向阳、黑土的生长条件下，独活中蛇床子素和二氢欧山芹醇当归酸酯的含量均较高。     

Osthole attenuates focal inflammatory reaction following permanent middle cerebral artery occlusion in rats

Osthole,a main active constituent from Cnidium monnieri(L.) Cusson,has been considered therapeutic agent in the treatment of ischemic stroke.This study was designed to investigate the effect of osthole on permanent middle cerebral artery occlusion(MCAO) in rats.Osthole was administrated by gavage to the normal and the MCAO rats.Rats were assessed for neurological deficit after 24 h following MCAO,then their brains were evaluated to determine the infarct area,and the mRNA and protein levels of some inflammatory factors were detected.It was found that MCAO animals pre-treated with osthole for 7 d showed significant improvement in all neurological tests compared with vehicle-treated MCAO groups.In addition,there was a significant decrease in infarct volume 24 h after occlusion in animals pre-treated with osthole versus the vehicle-treated MCAO group.MCAO also dramatically caused some inflammatory factors increase.However,pretreatment with osthole restored the mRNA and protein levels of these factors,including TNF-α,IL-1β,COX-2,iNOS of ischemic penumbra cortices,suggesting that osthole possessed the function of preventing brain against ischemic damage,while no significant difference was found in any of normal groups with or without osthole.The present study demonstrated that osthole may be a novel neuroprotective therapy in the treatment of focal ischemic stroke.     

复方雪莲风湿凝胶膏提取工艺研究

目的优选复方雪莲风湿凝胶膏提取工艺。方法采用正交试验设计,分别以蛇床子素含量、姜黄素含量和浸膏收率为指标,优化雪莲等7味药材的提取工艺;以没食子酸含量和浸膏收率为指标,优选土元和五倍子2味药的提取工艺。结果 7味药最佳提取工艺为10倍量80%乙醇,提取3次,每次1 h;2味药最佳提取工艺为10倍量水,提取3次,每次2.5 h。结论优选的提取工艺稳定、可行。     

苦柏妇炎栓精制工艺优选

目的:优选苦柏妇炎栓的精制工艺.方法:运用大孔吸附树脂法,以盐酸小檗碱、苦参碱、蛇床子素含量为指标,通过单因素试验筛选树脂型号及其精制工艺;采用HPLC测定指标成分含量.结果:采用HPD-400A型大孔吸附树脂,其最佳精制工艺为树脂(干重)-药材质量(1∶3),上样液质量浓度1.0 g·mL-1,上样液pH 8,洗脱流速1.5 BV·h-1,加5 BV水洗脱,之后分别用5 BV的20％乙醇和40％乙醇洗脱,合并乙醇洗脱液,减压回收,干燥,即得精制浸膏.结论:经HPD-400A型大孔吸附树脂精制后,栓剂载药量提高了4.3倍;优选的精制工艺稳定可行,可用于苦柏妇炎栓的工业化生产.     

HPLC法测定脊痛宁胶囊中蛇床子素的含量

目的建立脊痛宁胶囊中蛇床子素的含量测定方法。方法 HPLC法,色谱柱：Diamonsil（R）C18（250 mm×4.6 mm,5μm）;流动相：甲醇-水（68∶32）;流速：1.0 ml·min-1;检测波长：322 nm;柱温：40℃。结果蛇床子素在7.52~75.2μg·ml-1范围内线性关系良好,r=0.9999;回收率为101.30%,RSD为0.15%。结论本方法简便、准确,专属性强,测定结果重复性好,可用于脊痛宁中蛇床子素的质量控制。     

Osthole improves synaptic plasticity in the hippocampus and cognitive function of Alzheimer’s disease rats via regulating glutamate

Osthole, an effective monomer in Chinese medicinal herbs, can cross the blood-brain barrier and protect against brain injury, with few toxic effects. In this study, a rat model of Alzheimer’s disease was established after intracerebroventricular injection of β-amyloid peptide (25-35). Subsequently, the rats were intraperitoneally treated with osthole (12.5 or 25.0 mg/kg) for 14 successive days. Results showed that osthole treatment significantly improved cognitive impairment and protected hippocampal neurons of Alzheimer’s disease rats. Also, osthole treatment alleviated suppressed long-term potentiation in the hippocampus of Alzheimer’s disease rats. In these osthole-treated Alzheimer’s disease rats, the level of glutamate decreased, but there was no significant change in γ-amino-butyric acid. These experimental findings suggest that osthole can improve learning and memory impairment, and increase synaptic plasticity in Alzheimer’s disease rats. These effects of osthole may be because of its regulation of central glutamate and γ-amino-butyric acid levels.     

蛇床子素对大鼠心室肌细胞钠通道的影响

目的探讨蛇床子素对大鼠心室肌细胞钠离子通道电流(INa)的影响。方法采用Langendorff装置,恒压恒温灌流及酶解消化等方法对大鼠心肌细胞进行分离和处理。应用膜片钳技术,观察给予不同浓度蛇床子素(Ost)后钠离子通道电流特征的变化。结果 Ost(100μmol/L)能明显抑制钠电流,其作用呈浓度依赖性(500μmol/L几乎完全阻断)和时间依赖性(10 min左右抑制力达到最强)。100μmol/L和300μmol/L Ost使钠电流I-V曲线明显上移,峰值钠电流(-29.8±4.21)p A/p F分别降为(-20.1±3.7)p A/p F和(-17.7±5.7)p A/p F(P0.05),但激活电位和峰电位没有改变。对于钠通道失活曲线,不同浓度的Ost能使其向超级化方向移动,V1/2(空白,100μmol/L,300μmol/L)分别为(-81.10±0.35)、(-91.62±1.06)、(-100.60±0.21)m V(P0.01)。Ost还能显著延长钠通道失活后恢复时间,τ(空白,100μmol/L,300μmol/L)分别为(15.09±0.78)、(23.41±1.23)、(31.62±0.97)ms(P0.01)。结论 Ost对大鼠心室肌钠通道电流有明显的抑制作用。     

高速逆流色谱法分离制备独活中蛇床子素

目的:以独活粗提物为原料,利用高速逆流色谱法(HSCCC)分离制备高纯度蛇床子素。方法:利用超高效液相色谱(UPLC)分析并优化溶剂体系,选择正己烷-乙酸乙酯-甲醇-水(1.5∶2.5∶2∶1.5)为HSCCC溶剂系统,上相为固定相,下相为流动相,流速3 m L·min-1,主机转速850 r·min-1,进样量200 mg,检测波长323 nm。所接收馏分在50℃减压浓缩,得到蛇床子素单体,应用核磁共振氢谱、碳谱、质谱、红外光谱等进行鉴定,并用UPLC测定其纯度。结果:蛇床子素一次制备量为6.6mg,纯度达到97.1%。结论:高速逆流色谱技术操作简单,分离快速高效,一次制备量大,可以用于独活中高纯度蛇床子素的分离制备。     

蛇床子素对L02细胞的毒性作用及其机制研究

目的 探究蛇床子素（osthole,Ost）对L02细胞的毒性损伤和作用机制,为中药蛇床子及其制剂的安全合理应用提供实验依据。方法 以不同浓度Ost作用于L02细胞,MTT法检测细胞活性;乳酸脱氢酶（LDH）试剂盒测定细胞LDH释放率;Hoechst 33342染色法检测细胞核形态;Annexin V/PI双染法检测细胞凋亡;Western blot检测Bcl-2、Bax、pro-caspase-3、cleaved-caspase-3（p17）、p-Histon H3（Ser10）的表达。结果 L02细胞在Ost作用下活性下降,LDH释放率提高,且呈浓度依赖;Hoechst 33342染色荧光下可见细胞核皱缩碎裂;AnnexinV/PI双染法结果表明凋亡率随浓度提高而上升。与对照组比较,50,100,200 μmol·L^-1 Ost作用24 h后,Bcl-2、pro-caspase-3、p-Histon H3（Ser10）表达水平降低,Bax、cleaved-caspase-3表达水平升高。结论 Ost对L02细胞有毒性损伤作用,呈一定的时间和浓度依赖性,可促进细胞凋亡,抑制细胞增殖。     

蛇床子素在体外对人肝癌细胞增殖和侵袭的抑制作用

目的：研究蛇床子素在体外对人肝癌HepG2、SMMC-7721、Bel-7402细胞增殖和侵袭的抑制作用。方法：取人肝癌细胞进行复苏,传代,培养。实验分为空白组和给药组,给药组给予不同浓度梯度（50、100、150、200 μmol/L）蛇床子素药液处理。利用MTT法和细胞集落实验测定不同浓度蛇床子素体外抑制人肝癌细胞增殖作用;利用细胞划痕实验和Transwell小室实验测定不同浓度蛇床子素对人肝癌细胞的迁移和侵袭的抑制作用;利用蛋白质印迹（Western blot）法评价蛇床子素对人肝癌细胞中上皮细胞-间充质转化（EMT）相关蛋白E-cadherin和细胞迁移相关蛋白基质金属蛋白酶2（MMP-2）表达情况的影响。结果：蛇床子素能在体外抑制人肝癌细胞HepG2、SMMC-7721 和Bel-7402 的增殖,IC50 值分别为147.62、141.57、179.67 μmol/L,呈现一定的浓度依赖性。与空白组相比,给药组在蛇床子素50、75、100 μmol/L浓度条件下可以显著抑制人肝癌细胞增殖（P ＜0.05）。与空白组相比,给药组人肝癌细胞的迁移和侵袭能力显著降低（P ＜0.05）;给药组细胞MMP-2 蛋白的表达量降低（P ＜0.05）,E-cadherin蛋白的表达量增加（P ＜0.05）。结论：蛇床子素在体外对人肝癌细胞增殖和侵袭具有抑制作用,其抑制侵袭作用机制与下调MMP-2 蛋白和激活E-cadherin蛋白的表达有关。